Protein kinase targeted therapeutics

ABSTRACT

The present invention relates to compositions and methods useful in treating diseases and disorders related to protein kinases. In particular, the present invention relates to compositions and methods useful for targeting protein kinases related to mitogen activated protein kinase (MAPK) pathways (e.g., p38 MAPK, JNK, ERK, and upstream and downstream protein kinases) and/or casein kinase (CK) pathways (e.g., CK1δ, and upstream and downstream protein kinases), and diseases and disorders related to MAPK pathways (e.g., p38 MAPK, JNK, ERK, and upstream and downstream protein kinases) and/or CK pathways (e.g., CK1δ, and upstream and downstream protein kinases).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of pending U.S. patentapplication Ser. No. 11/833,152, filed Aug. 2, 2007, which claimspriority to expired U.S. Provisional Application No. 60/834,962, filedAug. 2, 2006, both of which are herein incorporated by reference intheir entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. NS047586 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful intreating diseases and disorders related to protein kinases. Inparticular, the present invention relates to compositions and methodsuseful for targeting protein kinases related to mitogen activatedprotein kinase (MAPK) pathways (e.g., p38 MAPK, JNK, ERK, and upstreamand downstream protein kinases) and/or casein kinase (CK) pathways(e.g., CK1δ, and upstream and downstream protein kinases), and diseasesand disorders related to MAPK pathways (e.g., p38 MAPK, JNK, ERK, andupstream and downstream protein kinases) and/or CK pathways (e.g., CK1δ,and upstream and downstream protein kinases).

BACKGROUND OF THE INVENTION

Intracellular protein kinases related to MAPK (e.g., p38 MAPK, JNK, ERK)and CK (e.g., CK1δ) are part of signal transduction pathways activatedby stress and represent desirable therapeutic targets, especially forinflammatory disorders and diseases (e.g., arthritis). Previous effortsto target (e.g., inhibit) intracellular protein kinases related to MAPK(e.g., p38 MAPK, JNK, ERK) and CK (e.g., CK1δ) have been met withfailure, in that previous inhibitors have had problems either directlyor indirectly related to a compound's molecular properties.

What are needed are compositions and methods that will effectivelytarget protein kinases related to MAPK (e.g., p38 MAPK, JNK, ERK) and/orCK (e.g., CK1δ), that have minimal or no toxicological impact to thesubject, and that can be useful as treatment and therapeutic options fordiseases and disorders related to MAPK pathways (e.g., p38 MAPK, JNK,ERK) and/or CK pathways (e.g., CK1δ).

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods useful intreating diseases and disorders related to protein kinases. Inparticular, the present invention relates to compositions and methodsuseful for targeting protein kinases related to mitogen activatedprotein kinase (MAPK) pathways (e.g., p38 MAPK, JNK, ERK, and upstreamand downstream protein kinases) and/or casein kinase (CK) pathways(e.g., CK1δ, and upstream and downstream protein kinases), and diseasesand disorders related to MAPK pathways (e.g., p38 MAPK, JNK, ERK, andupstream and downstream protein kinases) and/or CK pathways (e.g., CK1δ,and upstream and downstream protein kinases). Novel inhibitors ofprotein kinases related to MAPK (e.g., p38 MAPK, JNK, ERK, and upstreamand downstream protein kinases) and CK (e.g., CK1δ, and upstream anddownstream protein kinases) are presented, and the molecular propertiesof these inhibitors allow them to be orally bioavailable for use inanimal models and for human therapeutics alone or in combination withother therapeutic molecules.

In some embodiments, the compositions and methods of the presentinvention find use in research applications (e.g., drug screening,molecular and cellular based research, computer modeling scenarios).

In certain embodiments, the present invention provides compositionscomprising one or more anti-inflammatory agents and a compound describedby one or more of the following structures:

including salts and both R and S enantiomeric forms and racemic mixturesthereof, wherein R1, R2, R3, R4, and R6 are independently selected fromH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons; wherein R5 is H, CH3, a linear or branched, saturatedor unsaturated, a substituted or unsubstituted, aliphatic chain havingless than 5 carbons; wherein R7 is selected from the group consisting ofH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons.

In some embodiments, R3 is aromatic and R4 is a substituted orunsubstituted amine.

In some embodiments, the size of the R5 group is such that it does notsterically hinder the R4 group.

In some embodiments, the compound is one or more of:

In some embodiments, the one or more anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), anti-cytokines, imatinibmesylate, sorafenib, sunitinib malate, and anti-chemokines.

In some embodiments, the compound is an inhibitor of protein kinasesrelated to MAPK pathways (e.g., p38 MAPK, JNK, ERK, and upstream anddownstream protein kinases) and/or CK pathways (e.g., CK1δ, and upstreamand downstream protein kinases).

In certain embodiments, the present invention provides compositionscomprising one or more anti-inflammatory agents and a compound describedby the following formula:

including salts and both R and S enantiomeric forms and racemic mixturesthereof, wherein R1, R2, R3, R4, and R6 are independently selected fromH, C, CH, CH₂, CH₃,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons; wherein R5 is H, CH3, a linear or branched, saturatedor unsaturated, a substituted or unsubstituted, aliphatic chain havingless than 5 carbons; wherein R7 is selected from the group consisting ofH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons.

In some embodiments, R3 is aromatic and R4 is a substituted orunsubstituted amine.

In some embodiments, the size of the R5 group is such that it does notsterically hinder the R4 group.

In some embodiments, the compound is one or more of:

In some embodiments, the pharmaceutical composition comprises one ormore anti-inflammatory agents including non-steroidal anti-inflammatorydrugs (NSAIDs), anti-cytokines, imatinib mesylate, sorafenib, sunitinibmalate, and anti-chemokines.

In some embodiments, the compound is an inhibitor of protein kinasesrelated to MAPK pathways (e.g., p38 MAPK, JNK, ERK, and upstream anddownstream protein kinases) and/or CK pathways (e.g., CK1δ, and upstreamand downstream protein kinases).

In certain embodiments, the present invention provides methods fortreating a disorder selected from the group consisting of a skindisorder, an intestinal disorder, a lung disorder, interstitial cystitisof the bladder, coronary disease after ischemia-reperfusion injury,acute renal inflammation, bacterial otitis media, chorioretinal vasculardisease, a neurological disorder, aplastic anemia, bone marrow failuresyndrome, and cancer, comprising administering an effective amount of acompound to a subject suffering from the disorder, wherein the compoundcomprises the following formula:

including salts and both R and S enantiomeric forms and racemic mixturesthereof; wherein R1, R2, R3, R4, and R6 are independently selected fromH, C, CH, CH2, CH3.

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons; wherein R5 is H, CH3, a linear or branched, saturatedor unsaturated, a substituted or unsubstituted, aliphatic chain havingless than 5 carbons; wherein R7 is selected from the group consisting ofH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons.

In some embodiments, R3 is aromatic and R4 is a substituted orunsubstituted amine.

In some embodiments, the size of the R5 group is such that it does notsterically hinder the R4 group.

In some embodiments, the compound is one or more of:

In some embodiments, the compound is an inhibitor of protein kinasesrelated to MAPK pathways (e.g., p38 MAPK, JNK, ERK, and upstream anddownstream protein kinases) and/or CK pathways (e.g., CK1δ, and upstreamand downstream protein kinases).

In some embodiments, the methods result in prevention and/or a reductionof inflammation in the subject.

In some embodiments, the method further comprises administering anadditional agent for treating the disorder (e.g., an agent for treatingskin disorders, an agent for treating intestinal disorders, an agent fortreating lung disorders, an agent for treating neurological disorders,an agent for treating cancer (e.g., including metastasis), an agent fortreating interstitial cystitis of the bladder, an agent for treatingcoronary disease after ischemia-reperfusion injury, an agent fortreating acute renal inflammation, an agent for treating bacterialotitis media, an agent for treating chorioretinal vascular disease, anagent for treating aplastic anemia, and an agent for treating bonemarrow failure syndrome). In some embodiments, the agent is ananti-inflammatory agent such as non-steroidal anti-inflammatory drugs(NSAIDs), anti-cytokines, imatinib mesylate, sorafenib, sunitinibmalate, and anti-chemokines.

In some embodiments, the skin disorder is one or more of ichthyosisvulgaris, atopic dermatitis, psoriasis, eczema, allergic skin disease,and hypersensitivity reactions. In some embodiments, the intestinaldisorder is one or more of inflammatory bowel disease, Crohn's disease,ulcers, bacterial infections, hemorrhagic shock, diarrhea, colitis,pancreatitis. In some embodiments, the lung disorder is one or more ofacute lung injury after infection, sepsis, thrombin-induced lung injury,lung injury after reperfusion, chronic obstructive pulmonary disease,cystic fibrosis, inflammatory airway diseases. In some embodiments, theneurologic disorder is one or more of multiple sclerosis, Alzheimer'sdisease, vascular dementia, traumatic brain injury, ALS, Parkinson'sdisease, stroke, vasogenic brain edema, meningoencephalitis, cerebralhemorrhage, Guillain-Barre syndrome. In some embodiments, the cancer isprostate cancer, breast cancer, skin cancer, brain cancer, leukemia,lymphoma, and multiple myeloma. In some embodiments, the cancer is ametastasized cancer.

It should be understood that where different chemical constituents aredescribed for any particular location on a chemical scaffold describedherein, each of the possible combinations of constituents arecontemplated herein.

DEFINITIONS

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. In some embodiments of the present invention, test compoundsinclude antisense compounds.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like.

As used herein, the term “inflammatory condition” “inflammatorydisorder” or similar term refers to a condition wherein the organism'simmune cells are activated. Such a condition is characterized by aninflammatory response with pathologic sequelae. In some scenarios, thestate is characterized by infiltration of mononuclear cells,proliferation of fibroblasts and small blood vessels, increasedconnective tissue, and tissue destruction. Examples of inflammatorydiseases include, but are not limited to, arthritis (e.g.,osteoarthritis, rheumatoid arthritis, etc.), inflammatory bowel diseases(e.g., ulcerative colitis, Crohn's disease, etc.), inflammatory airwaydisorders and diseases (e.g., asthma, chronic obstructive pulmonarydisease, cystic fibrosis, etc), psoriasis, perfusion injury, restenosis,stenosis, myocardial injury, acute renal inflammation, bacterial otitismedia, acute ultraviolet irradiation (e.g., sunburn), endotoxic shock,and multiple sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of different routes of synthesis of the compounds,small molecules, and analogs thereof of the present invention.

FIG. 2 shows in vitro p38 MAPK and CK1δ inhibition of enzyme activity ina concentration dependent manner. In particular, in vitrophosphorylation of a standard protein or peptide substrate by purifiedhuman p38α MAPK and CK1δ was measured in the presence of 100 mM ATP, inthe absence or presence of increasing concentrations of MW01-2-069aSRM.Data are expressed as percent of the maximal enzyme activity, whereenzyme activity in the absence of MW01-2-069aSRM is taken as 100%.

FIG. 3 shows in vivo LPS-induced serum cytokine level inhibition in anacute inflammation mouse model with MW01-2-069aSRM. MW01-2-069aSRM (25mg/kg) was administered by oral gavage, then LPS (10 mg/kg) was giveni.p. 30 minutes later. After 6 hr, serum cytokine levels were measuredby ELISA. MW01-2-069aSRM was shown to be an effective p38a MAPKinhibitor.

FIG. 4 shows oral bioavailability, safety and metabolic stability withMW01-2-069aSRM. In particular, FIG. 4A shows plasma levels after oraladministration, FIG. 4B shows no liver histological toxicity screenafter chronic administration, and FIG. 4C shows metabolic stability inhuman liver microsome screen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods useful intreating diseases and disorders related to protein kinases. Inparticular, the present invention relates to compositions and methodsuseful for targeting protein kinases related to mitogen activatedprotein kinase (MAPK) pathways (e.g., p38 MAPK, JNK, ERK, and upstreamand downstream protein kinases) and/or CK pathways (e.g., CK1δ, andupstream and downstream protein kinases), and diseases and disordersrelated to MAPK pathways (e.g., p38 MAPK, JNK, ERK, and upstream anddownstream protein kinases) and/or CK pathways (e.g., CK1δ, and upstreamand downstream protein kinases).

The mitogen activated protein kinases (MAPKs) are serine-threonineprotein kinases that integrate and process extracellular stimuli througha series of intracellular signaling complexes and phosphorylationcascades that lead to stimulus-specific responses (see, e.g., Kyriakis JM and Avruch J (2001) Physiol Rev 81: 808-869; herein incorporated byreference in its entirety). In a simplest form, the MAPK cascadeconsists of a three-tiered series of protein kinases: a MAPK (ERKs,JNKs, p38s) and two upstream components (a MAPK kinase and a MAPKKkinase) that activate the MAPKs by a series of activatingphosphorylations (see, e.g., Bonni A, et al., Science. 1999 Nov. 12;286(5443):1358-62; Chadee D N, Mol Cell Biol. 2002 February;22(3):737-49; Chang L, et al., Nature 2001 Mar. 1; 410(6824):37-40; ChenY R, et al., Gene ther mol biol Vol 4, 83-98 Dec. 1999; Hazzalin C A, etal., Nat Rev Mol Cell Biol. 2002 January; 3(1):30-40; Kato Y, et al.,EMBO J. 1997 Dec. 1; 16(23):7054-66; Kiefer F, et al., EMBO J. 1996 Dec.16; 15(24):7013-25; Pearson G, et al., J Biol. Chem. 2001 Mar. 16;276(11):7927-31; Weston C R, et al., Science. 2002 Jun. 28;296(5577):2345-7; each herein incorporated by reference in theirentireties). The activated MAPK can phosphorylate a number ofsubstrates, including transcription factors, which then lead tostimulus-specific responses. This description of the MAPK cascades aslinear, isolated pathways is understood to be oversimplified, becausethe MAPK pathways can interact with and be influenced by other signalingpathways, by interactions with scaffolding proteins, and by specificlocalizations within cells.

Protein kinases related to the MAPK pathway (e.g., p38 MAPK, JNK, ERK)are therapeutic targets for both inflammatory diseases (see, e.g.,Kaminska B (2005) Biochim Biophys Acta 1754: 253-262; Kumar S, et al.,Nature Rev Drug Discovery 2: 717-726; Saklatvala J (2004) Curr OpinPharmacol 4: 372-377; each herein incorporated by reference in theirentireties) and cancer (see, e.g., Engelberg D (2004) Sem Cancer Biol14: 271-282; Platanias L C (2003) Blood 101: 4667-4679; Rennefahrt U, etal., (2005) Cancer Lett 217: 1-9; each herein incorporated by referencein their entireties). The p38 MAPK family consists of at least fourisoforms, p38α, β (and P2), δ, γ, which are encoded by separate genes,expressed in different tissues and may have distinct functions.

In terms of p38 MAPK's role in inflammatory diseases, activation of p38MAPK has been shown to regulate gene expression and lead to increasedproduction of proinflammatory cytokines (see, e.g., Schieven G L (2005)Curr Topics Med Chem 5: 921-928; herein incorporated by reference in itsentirety). The mechanisms by which p38 MAPK stimulates proinflammatorycytokine production include phosphorylation and activation oftranscription factors, some of which can increase transcription ofinflammatory cytokine genes; regulation of cytokine mRNA stability andtranslation; and regulation of transcriptional activation of certaincytokines. Thus, p38 MAPK can modulate a number of different signalingevents that can converge on proinflammatory cytokine up-regulation.

p38 MAPK has been found to be a component of tumor suppressor pathwaysunder some conditions and as a pro-oncogenic component under otherconditions (see, e.g., Engelberg D (2004) Sem Cancer Biol 14: 271-282;Rennefahrt U, et al., (2005) Cancer Lett 217: 1-9; each hereinincorporated by reference in their entireties). The kinase has beenshown to be involved in cell growth, differentiation, cell cyclecontrol, and apoptosis. In certain cancers, p38 MAPK is activated andmediates cell proliferation. In addition, p38 MAPK is activated inresponse to environmental stresses and damaging agents, such as UVirradiation, which can induce tumors (see, e.g., Jinlian L (2007) JBiomed Sci 14: 303-312; herein incorporated by reference in itsentirety).

Because of its importance in modulating proinflammatory cytokineproduction, p38 MAPK is a compelling therapeutic targets for smallmolecule development against inflammatory diseases characterized byelevated levels of proinflammatory cytokines. In addition, because ofthe increasing awareness of the pro-oncogenic potential of p38 MAPK andits observed effects in mediating a number of cellular processesimportant for cancer onset and progression, targeting p38 MAPK is atherapeutic option for treating cancer, especially for hematologicmalignancies (see, e.g. Platanias L C (2003) Blood 101: 4667-4679;herein incorporated by reference in its entirety). The present inventionis not limited to a particular mechanism. Indeed, an understanding ofthe mechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that the inhibition of p38 MAPK is auseful therapy for diseases and/or disorders where p38 MAPK isover-expressed or over-activated.

CK protein kinases (e.g., CK1δ) are also serine-threonine kinases. TheCK protein kinases (e.g., CK1δ) are highly conserved within their kinasedomains, but differ in their N- and C-terminal non-catalytic domains(see, e.g., Knippschild U, et al., 2005, Cellular Signalling 17:675-689; Knippschild U, et al., 2005, Onkologie 28: 508-514; each hereinincorporated by reference in their entireties). The functional activityof the CK protein kinases (e.g., CK1δ) in cells can be regulated by avariety of means, including modulation of CK protein kinase (e.g., CK1δ)expression, activity, and subcellular localization. A large number ofsubstrates for CK protein kinases (e.g., CK1δ) have been discovered,including enzymes, transcription factors, viral oncogenes, cytoskeletalproteins, membrane associated proteins, and receptors. The diversity ofCK (e.g., CK1δ) substrates indicates that the enzyme is involved inregulation of diverse cellular processes. Some examples of functionsascribed to CK protein kinases (e.g., CK1δ) include, but are not limitedto, regulation of circadian rhythms through phosphorylation andmodulation of the functions of specific clock proteins; involvement incancer development through phosphorylation of the tumor suppressor p53and the cellular oncogene Mdm2, resulting in uncontrolled cell growth,and through elevated CK (e.g., CK1δ) levels and/or activity in a varietyof cancers; inhibition of apoptosis induced through different pathways,including mediating resistance of tumor cells to caspase action andinterfering with retinoic acid receptor mediated apoptosis; modulationof Wnt-signaling pathways; and overexpression and involvement inneurodegenerative diseases. The present invention is not limited to aparticular mechanism. Indeed, an understanding of the mechanism is notnecessary to practice the present invention. Nonetheless, it iscontemplated that CK protein kinases (e.g., CK1δ) are linked to varioustypes of diseases such as cancer, inflammatory diseases, and otherdisorders. In addition, it is contemplated that the inhibition of CKprotein kinases (e.g., CK1δ) is a useful therapy for diseases and/ordisorders where CK protein kinases (e.g., CK1δ) are over-expressed orover-activated.

In some embodiments, the compositions and methods of the presentinvention are useful in prevention and/or treatment of diseases and/ordisorders where protein kinases related to the MAPK pathway (e.g., p38MAPK, JNK, ERK) and/or the CK pathway (e.g., CK1δ) are over-expressed orover-activated. The present invention is not limited to treating certaintypes of diseases and/or disorders where protein kinases related to theMAPK pathway (e.g., p38 MAPK, JNK, ERK) and/or the CK pathway (e.g.,CK1δ) are over-expressed or over-activated. In some embodiments, thecompositions and methods of the present invention are useful inprevention and/or treatment of inflammatory diseases that include, butare not limited to, arthritis (e.g., osteoarthritis, rheumatoidarthritis, etc.), inflammatory bowel diseases (e.g., ulcerative colitis,Crohn's disease, etc.), inflammatory airway disorders and diseases(e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis,etc), psoriasis, perfusion injury, restenosis, stenosis, myocardialinjury, acute renal inflammation, bacterial otitis media, acuteultraviolet irradiation (e.g., sunburn) and endotoxic shock. In someembodiments, the compositions and methods of the present invention areuseful in prevention and/or treatment of Alzheimers disease and relateddisorders, presenile and senile forms; amyloid angiopathy; mildcognitive impairment; Alzheimer's disease-related dementia (e.g.,vascular dementia or Alzheimer dementia); AIDS related dementia,tauopathies (e.g., argyrophilic grain dementia, corticobasaldegeneration, dementia pugilistica, diffuse neurofibrillary tangles withcalcification, frontotemporal dementia with parkinsonism, Prion-relateddisease, Hallervorden-Spatz disease, myotonic dystrophy, Niemann-Pickdisease type C, non-Guamanian Motor Neuron disease with neurofibrillarytangles, Pick's disease, postencephalitic parkinsonism, cerebral amyloidangiopathy, progressive subcortical gliosis, progressive supranuclearpalsy, subacute sclerosing panencephalitis, and tangle only dementia),alpha-synucleinopathy (e.g., dementia with Lewy bodies, multiple systematrophy with glial cytoplasmic inclusions), multiple system atrophies,Shy-Drager syndrome, spinocerebellar ataxia (e.g., DRPLA orMachado-Joseph Disease); striatonigral degeneration,olivopontocerebellar atrophy, neurodegeneration with brain ironaccumulation type I, olfactory dysfunction, and amyotrophic lateralsclerosis); Parkinson's disease (e.g., familial or non-familial);Amyotrophic Lateral Sclerosis; Spastic paraplegia (e.g., associated withdefective function of chaperones and/or triple A proteins); Huntington'sDisease, spinocerebellar ataxia, Freidrich's Ataxia; cerebrovasculardiseases including stroke, hypoxia, ischemia, infarction, intracerebralhemorrhage; traumatic brain injury; Down's syndrome; head trauma withpost-traumatic accumulation of amyloid beta peptide; Familial BritishDementia; Familial Danish Dementia; Presenile Dementia with SpasticAtaxia; Cerebral Amyloid Angiopathy, British Type; Presenile DementiaWith Spastic Ataxia Cerebral Amyloid Angiopathy, Danish Type; Familialencephalopathy with neuroserpin inclusion bodies (FENIB); AmyloidPolyneuropathy (e.g., senile amyloid polyneuropathy or systemicAmyloidosis); Inclusion Body myositis due to amyloid beta peptide;Familial and Finnish Type Amyloidosis; Systemic amyloidosis associatedwith multiple myeloma; Familial Mediterranean Fever; multiple sclerosis,optic neuritis; Guillain-Barre Syndrome; chronic inflammatorydemyelinating polyneuropathy; chronic infections and inflammations;acute disseminated encephalomyelitis (ADEM); autoimmune inner eardisease (AIED); diabetes; pancreatitis; gout; artherioscierosis,inflammatory aortic aneurysm; glomerulonephritis; sacoidosis cancer;restenosis; rheumatic fever; systemic lupus erythematosus; Reiter'ssyndrome; psoriatic arthritis; ankylosing spondylitis; coxarthritis;pelvic inflammatory disease; osteomyelitis; adhesive capsulitis;oligoarthritis; periarthritis; polyarthritis; Still's disease;synovitis; inflammatory dermatosis; and, wound healing. In someembodiments, the compositions and methods of the present invention areuseful in prevention and/or treatment of skin disorders: includingichthyosis vulgaris, atopic dermatitis, psoriasis, eczema, allergic skindisease, and hypersensitivity reactions; intestinal disorders: includinginflammatory bowel disease, Crohn's disease, ulcers, bacterialinfections, hemorrhagic shock, diarrhea, colitis, pancreatitis; lungdisorders: including acute lung injury after infection, sepsis,thrombin-induced lung injury, lung injury after reperfusion, chronicobstructive pulmonary disease, cystic fibrosis, inflammatory airwaydiseases; interstitial cystitis of the bladder; coronary disease afterischemia-reperfusion injury; acute renal inflammation; bacterial otitismedia; chorioretinal vascular disease; neurologic disorders: includingmultiple sclerosis, Alzheimer's disease, vascular dementia, traumaticbrain injury, ALS, Parkinson's disease, stroke, vasogenic brain edema,meningoencephalitis, cerebral hemorrhage, Guillain-Barre syndrome;aplastic anemia; bone marrow failure syndrome; and cancers, includingorgan cancers such as prostate, breast, skin, brain, and hematopoieticmalignancies including leukemias, lymphomas, and multiple myeloma. Oneskilled in the art will recognize a myriad of other disorders anddiseases that would be equally preventable and/or treatable by thecompositions and methods of the present invention.

In some embodiments, the present invention provides compositionscomprising compounds, small molecules and/or analogs thereof useful ininhibiting protein kinase activity related to the MAPK pathway (e.g.,p38 MAPK, JNK, ERK, and upstream and downstream protein kinases) and/orthe CK pathway (e.g., CK1δ, and upstream and downstream proteinkinases). The present invention is not limited to particular typesand/or kinds of compounds, small molecules and/or analogs thereof thatare useful in inhibiting protein kinase activity related to the MAPKpathway (e.g., p38 MAPK, JNK, ERK, and upstream and downstream proteinkinases) and/or the CK pathway (e.g., CK1δ, and upstream and downstreamprotein kinases). In some embodiments, the compounds, small moleculesand/or analogs thereof that are useful in inhibiting protein kinaseactivity related to the MAPK pathway (e.g., p38 MAPK, JNK, ERK, andupstream and downstream protein kinases) and/or the CK pathway (e.g.,CK1δ, and upstream and downstream protein kinases) do not inhibitrelated protein kinases such as DAPK, PKC, PKA, or MLCK (e.g., asdetermined in a standard activity assay in vitro or in vivo).

In some embodiments, the compounds, small molecules and/or analogsthereof that are useful in inhibiting protein kinase activity related tothe MAPK pathway (e.g., p38 MAPK, JNK, ERK, and upstream and downstreamprotein kinases) and/or the CK pathway (e.g., CK1δ, and upstream anddownstream protein kinases) are described by the following structures:

including salts and both R and S enantiomeric forms and racemic mixturesthereof, wherein R1, R2, R3, R4, and R6 are independently selected fromH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons; wherein R5 is H, CH3, a linear or branched, saturatedor unsaturated, a substituted or unsubstituted, aliphatic chain havingless than 5 carbons; wherein R7 is selected from the group consisting ofH, C, CH, CH2, CH3,

and a substituted or unsubstituted chemical moiety comprising at leastone of the following: alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl,sulfenyl, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,ureido, cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,carboxyl, carbonyl, carbamoyl, carboxamide, hydrogen, amino, nitrogen,pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine,pyrimidine, quinazoline, pyridazine, piperazine, cinnoline,cycloaliphatic subgroup, ester, ether, sulfur, phosphorous, oxygen, anaromatic ring, a non-aromatic ring, a linear or branched, saturated orunsaturated, a substituted or unsubstituted, aliphatic chain having atleast 2 carbons.

In some embodiments, R3 is aromatic and R4 is a substituted orunsubstituted amine.

In some embodiments, the size of the R5 group is such that it does notsterically hinder the R4 group.

In some embodiments, the compound is one or more of:

The present invention is not limited to particular synthesis schemes forgenerating the compounds of the present invention. In some embodiments,the synthesis schemes depicted in FIG. 1 are used to generate thecompounds of the present invention. In some embodiments, the synthesisschemes described in WO 2006/050389, herein incorporated by reference inits entirety, are utilized for generating the compounds of the presentinvention

In some embodiments, as shown in FIG. 2, experiments conducted duringthe course of development of embodiments of the present inventiondemonstrated that MW01-2-069aSRM or analogs thereof inhibit proteinkinase activity. As shown in FIG. 2, MW01-2-069aSRM was shown to inhibitin vitro p38 MAPK and CK1δ enzyme activity in a concentration dependentmanner. As shown in FIG. 3, MW01-2-069aSRM was shown to inhibitLPS-induced serum cytokine levels in vivo in an acute inflammation mousemodel. MW01-2-069aSRM (25 mg/kg) was administered by oral gavage, thenLPS (10 mg/kg) was given i.p. 30 minutes later. After 6 hr, serumcytokine levels were measured by ELISA. MW01-2-069aSRM was shown to bean effective p38α MAPK inhibitor. As shown in FIG. 4, oralbioavailability, safety and metabolic stability is demonstrated forMW01-2-069aSRM. MW01-2-069aSRM exemplifies the activity and propertiesof the classes of compounds described herein.

In some embodiments, the compounds, small molecules and/or analogsthereof that are useful in inhibiting protein kinase activity related tothe MAPK pathway (e.g., p38 MAPK, JNK, ERK) and/or the CK pathway (e.g.,CK1δ) are administered to subjects to prevent an inflammatory disorderor disease. In some embodiments, the compounds, small molecules oranalogs thereof are administered to subjects as a therapeutic to treat asubject with an inflammatory disorder or disease. In some embodiments,the compounds, small molecules or analogs thereof are administered tonon-human animals useful as test subjects. In some embodiments, thecompounds, small molecules or analogs thereof are administered tohumans. In some embodiments, the compounds, small molecules or analogsthereof are administered to subjects in combination with othertherapies.

In some embodiments, the compounds, small molecules or analogs thereofare administered to subjects in combination with anti-inflammatoryagents. The present invention is not limited to particular types oramounts of anti-inflammatory agents. In some embodiments,anti-inflammatory agents include, but are not limited to, non-steroidalanti-inflammatory drugs (NSAIDs), anti-cytokines, anti-chemokines,imatinib mesylate, sorafenib, sunitinib malate, and other smallmolecules, drugs, compounds know in the art to inhibit inflammation oran inflammatory response.

Additionally, any one or more of the compounds, small molecules oranalogs thereof can be used in combination with at least one othertherapeutic agent (e.g., potassium channel openers, calcium channelblockers, sodium hydrogen exchanger inhibitors, antiarrhythmic agents,antiatherosclerotic agents, anticoagulants, antithrombotic agents,prothrombolytic agents, fibrinogen antagonists, diuretics,antihypertensive agents, ATPase inhibitors, mineralocorticoid receptorantagonists, phosphodiesterase inhibitors, antidiabetic agents,anti-inflammatory agents, antioxidants, angiogenesis modulators,antiosteoporosis agents, hormone replacement therapies, hormone receptormodulators, oral contraceptives, antiobesity agents, antidepressants,antianxiety agents, antipsychotic agents, antiproliferative agents,antitumor agents, antiulcer and gastroesophageal reflux disease agents,growth hormone agents and/or growth hormone secretagogues, thyroidmimetics, anti-infective agents, antiviral agents, antibacterial agents,antifungal agents, cholesterol/lipid lowering agents and lipid profiletherapies, and agents that mimic ischemic preconditioning and/ormyocardial stunning, antiatherosclerotic agents, anticoagulants,antithrombotic agents, antihypertensive agents, antidiabetic agents, andantihypertensive agents selected from ACE inhibitors, AT-1 receptorantagonists, ET receptor antagonists, dual ET/AII receptor antagonists,and vasopepsidase inhibitors, or an antiplatelet agent selected fromGPIIb/IIIa blockers, P2Y₁ and P2Y₁₂ antagonists, thromboxane receptorantagonists, and aspirin) in along with a pharmaceutically-acceptablecarrier or diluent in a pharmaceutical composition.

In certain embodiments, one or more of the compounds, small molecules oranalogs thereof can be used in combination with a therapeutic agentselected from the group consisting of potassium channel openers, calciumchannel blockers, sodium hydrogen exchanger inhibitors, antiarrhythmicagents (e.g., sotalol, dofetilide, amiodarone, azimilide, ibutilide,ditiazem, verapamil), antiatherosclerotic agents, anticoagulants,antithrombotic agents, prothrombolytic agents, fibrinogen antagonists,diuretics, antihypertensive agents (e.g., captopril, lisinopril,zofenopril, ramipril, fosinopril, enalapril, ceranopril, cilazopril,delapril, pentopril, quinapril, omapatrilat, gemopatrilat, losartan,irbesartan, valsartan, sitaxsentan, atrsentan), ATPase inhibitors,mineralocorticoid receptor antagonists, phosphodiesterase inhibitors,antidiabetic agents, anti-inflammatory agents, antioxidants,angiogenesis modulators, antiosteoporosis agents, hormone replacementtherapies, hormone receptor modulators, oral contraceptives, antiobesityagents, antidepressants, antianxiety agents, antipsychotic agents,antiproliferative agents, antitumor agents, antiulcer andgastroesophageal reflux disease agents, growth hormone agents and/orgrowth hormone secretagogues, thyroid mimetics, anti-infective agents,antiviral agents, antibacterial agents, antifungal agents,cholesterol/lipid lowering agents and lipid profile therapies, andagents that mimic ischemic preconditioning and/or myocardial stunning,antiatherosclerotic agents, anticoagulants, antithrombotic agents,antihypertensive agents, antidiabetic agents, and antihypertensiveagents including, but not limited to, ACE inhibitors, AT-1 receptorantagonists, ET receptor antagonists, dual ET/AII receptor antagonists,and vasopepsidase inhibitors, or an antiplatelet agent (plateletinhibitor) comprising GPIIb/IIIa blockers, P2Y₁ and P2Y₁₂ antagonists,thromboxane receptor antagonists, abciximab, eptifibatide, tirofiban,clopidogrel, ticlopidine, CS-747, ifetroban, and aspirin. In certaininstances, the therapeutic agent is propafenone, propranolol; sotalol,dofetilide, amiodarone, azimilide, ibutilide, ditiazem, verapamil,captopril, lisinopril, zofenopril, ramipril, fosinopril, enalapril,eranopril, cilazopril, delapril, pentopril, quinapril, omapatrilat,gemopatrilat, losartan, irbesartan, valsartan, sitaxsentan, atrsentan;verapamil, nifedipine, diltiazem, amlodipine and mybefradil, digitalis,ouabain, chlorothiazide, hydrochlorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichloromethiazide, polythiazide, benzthiazide, ethacrynic acidtricrynafen, chlorthalidone, furosemide, musolimine, bumetanide,triamtrenene, amiloride, spironolatone, aplirinone, dipyridamole,cilostazol, sildenafil, ifetroban, picotamide, ketanserin, clopidogrel,picotamide, rosuvastaitin, atavastatin visastatin, questran, CP-529414,lovenox, enoxaparain dalteparinnadolol, carvedilol, albuterol,terbutaline, formoterol, salmeterol, bitolterol, pilbuterol, fenoterol,ipratropium bromide, metformin, acarbose, repaglinide, glimpepiride,glyburide, glyburide, glipizide, glucovance, troglitazone,rosiglitazone, pioglitazone, GLP-1, nefazodone, sertraline, diazepam,lorazepam, buspirone, hydroxyzine pamoate, acarbose, endostatin,probucol, BO-653, Vitamin A, Vitamin E, AGI-1067, alendronate,raloxifene, orlistate, cyclosperine A, paclitaxel, FK506, adriamycin,famotidine, rapitidine, ompeprazole, estrogen, estradiol, dipyridamole,cilostazol, sildenafil, ketanserin, taxol, cisplatin, paclitaxel,adriamycin, epothilones, carboplatin, cromolyn, nedocromil,theophylline, zileuton, zafirlukast, monteleukast, pranleukast,beclomethasone, triamcinolone, budesonide, fluticasone, flunisolidemprednisone; dexamethasone, etanercept, aspirin, indomethacin,pravastatin, simvastatin, atorvastatin, fluvastatin, cerivastatin,AZ4522, itavastatin, ZD-4522, rosuvastatin, atavastatin, visastatin,abciximab, eptifibatide, tirofiban, clopidogrel, ticlopidine, CS-747,ifetroban, aspirin; cariporide, streptokinase, reteplase, activase,lanoteplase, urokinase, prourokinase, tenecteplase, lanoteplase,anistreplase, eminase, lepirudin, argatroban, XR-330, T686,anti-α-2-antiplasmin antibody, or doesdipyridanmol.

In some embodiments, the compounds, small molecules or analogs thereofare useful in research studies, such as studying pathways associatedwith serine/threonine protein kinases using in vivo or in vitro assays.

In some embodiments the present invention provides methods of storageand administration of the antagonist, agent, compound, or drug in asuitable environment (e.g. buffer system, adjuvants, etc.) in order tomaintain the efficacy and potency of the agent, compound, or drug suchthat its usefulness in treating a disease and/or disorder relating tothe MAPK pathway (e.g., p38 MAPK, JNK, ERK) and/or the CK pathway (e.g.,CK1δ) is maximized. For example, small molecules or compounds benefitfrom a storage environment free of enzymes or compounds that could causedegradation of the small molecule, chemical, or compound.

In some embodiments, it is contemplated that the antagonist, agent,compound, or drug is administered to the individual as part of apharmaceutical or physiological composition for treating a diseaseand/or disorder relating to the MAPK pathway (e.g., p38 MAPK, JNK, ERK)and/or the CK pathway (e.g., CK1δ). Such a composition can comprise asmall molecule and a physiologically acceptable carrier. Pharmaceuticalcompositions for co-therapy can further comprise one or more additionaltherapeutic agents. The formulation of a pharmaceutical composition canvary according to the route of administration selected (e.g., solution,emulsion, capsule). Suitable pharmaceutical carriers can contain inertingredients that do not interact with the small molecule function and/oradditional therapeutic agent(s). Standard pharmaceutical formulationtechniques can be employed, such as those described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theparticular co-therapeutic agent selected for administration with a MAPKpathway (e.g., p38 MAPK, JNK, ERK) inhibitor and/or the CK pathway(e.g., CK1δ) inhibitor. The type inhibitor will depend on the type andseverity of the disease or disorder being treated as well as thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs.

In some embodiments the therapeutic agent is administered by anysuitable route, including, for example, orally (e.g., in capsules,suspensions or tablets) or by parenteral administration. Parenteraladministration can include, for example, intramuscular, intravenous,intraarticular, subcutaneous, or intraperitoneal administration. In someembodiments, the method of administration of the therapeutic agent is bydirect injection into, or adjacent to, the sire of inflammation. Thesmall molecule therapeutic agent can also be administered transdermally,topically, by inhalation (e.g., intrabronchial, intranasal, oralinhalation or intranasal drops) or rectally. Administration can be localor systemic as indicated. The preferred mode of administration can varydepending upon the particular agent chosen. A timed-release,subcutaneous mode of administration is also contemplated. For example, atherapeutic agent is inserted under the skin either by injection, and/orby placing a solid support that has been previously impregnated or whichcontains (e.g., a capsule) the therapeutic agent, under the skin. Aneffective amount of the therapeutic agent is then released over time(e.g., days, weeks, months, and the like) such that the subject is notrequired to have a therapeutic agent administered on a daily basis.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion (see, e.g., WO 2006/050389, hereinincorporated by reference in its entirety). In other embodiments, theactive ingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers and are made bycompressing or molding the respective agents. In some embodiments,compressed tablets are prepared by compressing in a suitable machine theactive ingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent.

In some embodiments of the present invention, the compounds, smallmolecules and analogs thereof of the present invention, and otherpotentially useful compounds, are screened for their binding affinityto, for example, protein kinases related to the MAPK pathway (e.g., p38MAPK, JNK, ERK) and/or the CK pathway (e.g., CK1δ). A number of suitablescreens for measuring the binding affinity of drugs and other smallmolecules to receptors are known in the art. In some embodiments,binding affinity screens are conducted in in vitro systems. In otherembodiments, these screens are conducted in in vivo or ex vivo systems.

Additional embodiments are directed to measuring expression levels(e.g., intracellular) of protein kinases related to the MAPK pathway(e.g., p38 MAPK, JNK, ERK) and/or the CK pathway (e.g., CK1δ) in cellsand/or tissues to measure the effectiveness of particular contemplatedmethods and compounds of the present invention.

In some embodiments, structure-based virtual screening methodologies arecontemplated for predicting the binding affinity of compounds of thepresent invention with protein kinases related to the MAPK pathway(e.g., p38 MAPK, JNK, ERK) and/or the CK pathway (e.g., CK1δ). In someembodiments, compound structures are predicted from a molecular modelingsoftware (e.g., MacroModel).

Any suitable assay that allows for a measurement of the rate of bindingor the affinity of an exemplary compound of the present invention toprotein kinases related to the MAPK pathway (e.g., p38 MAPK, JNK, ERK)and/or the CK pathway (e.g., CK1δ) may be utilized. Examples include,but are not limited to, competition binding using an exemplary compound,surface plasma resonance (SPR) and radio-immunopreciptiation assays(Lowman et al., J. Biol. Chem. 266:10982 [1991]). Surface PlasmonResonance techniques involve a surface coated with a thin film of aconductive metal, such as gold, silver, chrome or aluminum, in whichelectromagnetic waves, called Surface Plasmons, can be induced by a beamof light incident on the metal glass interface at a specific anglecalled the Surface Plasmon Resonance angle. Modulation of the refractiveindex of the interfacial region between the solution and the metalsurface following binding of the captured macromolecules causes a changein the SPR angle which can either be measured directly or which causesthe amount of light reflected from the underside of the metal surface tochange. Such changes can be directly related to the mass and otheroptical properties of the molecules binding to the SPR device surface.Several biosensor systems based on such principles have been disclosed(See e.g., WO 90/05305). There are also several commercially availableSPR biosensors (e.g., BiaCore, Uppsala, Sweden).

In some embodiments, compounds are screened in cell culture or in vivo(e.g., non-human or human mammals) for an ability to modulate (e.g.,alter activity) protein kinases related to the MAPK pathway (e.g., p38MAPK, JNK, ERK) and/or the CK pathway (e.g., CK1δ). Any suitable assaymay be utilized, including, but not limited to, cell proliferationassays (Commercially available from, e.g., Promega, Madison, Wis. andStratagene, La Jolla, Calif.) and cell based dimerization assays. (Seee.g., Fuh et al., Science, 256:1677 [1992]; Colosi et al., J. Biol.Chem., 268:12617 [1993]). Additional assay formats that find use withthe present invention include, but are not limited to, assays formeasuring cellular MAPK (e.g., p38 MAPK) and/or the CK pathway (e.g.,CK1δ) levels.

The present invention also provides methods of modifying andderivatizing the compositions of the present invention to increasedesirable properties (e.g., binding affinity, activity, and the like),or to minimize undesirable properties (e.g., nonspecific reactivity,toxicity, and the like). The principles of chemical derivatization arewell understood. In some embodiments, iterative design and chemicalsynthesis approaches are used to produce a library of derivatized childcompounds from a parent compound. In other embodiments, rational designmethods are used to predict and model in silico ligand-receptorinteractions prior to confirming results by routine experimentation.

EXAMPLES Example I Compound Synthesis Schemes

FIG. 1 shows different exemplary routes of synthesis for the compounds,small molecules, and analogs thereof of the present invention.

1-Phenyl-2-(pyridin-4-yl)ethanone: (2) To a stirred solution of lithiumdiisopropylamide (1.5 eq) in THF at −78° C. a solution of 4-picoline (1eq) in THF is added slowly. After 15 min, a solution ofN-methoxy-N-methylbenzamide (1.2 eq) in THF is added. The reactionmixture is allowed to warm to ambient temperature. The formation ofproduct is monitored by HPLC. The reaction mixture is poured into asaturated aqueous sodium bicarbonate solution, then extracted withethylacetate. The organic phase is washed with brine, dried overanhydrous magnesium sulphate and evaporated under reduced pressure togive the crude product. The crude product is then purified by flashcolumn chromatography (25-100% EtOAc-hexane) to give the product ketone2. Purity>90%, ESI m/z 198.18 (MH⁺).

Ethyl 4-oxo-4-phenyl-3-(pyridin-4-yl)butanoate: (3) Sodium hydride (1.1eq) and anhydrous THF are combined under argon at 0° C., and a solutionof compound (2) (1 eq) in anhydrous THF is added and stirred. A solutionof ethyl bromoacetate (1.5 eq) in anhydrous THF is added, the reactionmixture stirred at ambient temperature, and the reaction progressmonitored using HPLC. The reaction mixture is slowly poured into water,saturated sodium bicarbonate added, and the resulting solution extractedwith ethyl acetate. The organic extracts are dried over anhydrous sodiumsulphate and then concentrated under reduced pressure to give the crudeproduct mixture. Purification by flash column chromatography(hexane:ethyl acetate 70:30) gives the product keto-ester 3. Purity>90%,ESI m/z 284.75 (MH⁺).

6-Phenyl-5-(pyridin-4-yl)-4,5-dihydropyridazin-3(2H)-one: (4) Thecompound (3) (1 eq) is mixed in ethanol, hydrazine (2 eq) added slowly,and the resulting solution is heated under reflux with continuousstirring until completion (monitored by HPLC). The reaction mixture iscooled to ambient temperature and solvent evaporated in vacuo. Ethylacetate is added, followed by ether, and the solution rotary evaporatedto produce a foam-like solid, which can be used for the synthesis ofnext step without further purification. EI m/z 251.1 (M⁺).

6-Phenyl-5-(pyridin-4-yl)pyridazin-3-ol: (5) Compound (4) in 5 ml ofacetic acid is mixed gradually with. 1.5 eq bromine (in acetic acid),refluxed at 70° C., and the reaction progress monitored by HPLC untilall starting material disappears. The mixture is cooled to ambienttemperature, poured unto crushed ice to quench the reaction, the pH ofthe aqueous reaction mixture adjusted to pH 6 with 0.2 N NaOH, and themixture extracted with ethyl acetate. The aqueous phase is dried invacuo and the residue extracted with ethyl acetate and dichloromethane.Final purification by flash column chromatography over silica gelcolumn, eluting with dichloromethane and MeOH (5%), affords compound 5.Purity>90%, ESI m/z 250.20 (MH⁺).

6-Chloro-3-phenyl-4-(pyridin-4-yl)Pyridazine: (6) Compound 5 (1 eq) andPOCl₃ (40 mmol, 10 eq) are mixed, stirred at 90° C., and the reactionmonitored by HPLC. The resultant mixture is poured unto crushed ice andmixed for 30 min to decompose POCl₃, the pH mixture adjusted 7, and themixture is extracted with ethyl acetate and dichloromethane. The organicextracts are combined and dried over anhydrous magnesium sulfate, thesolvent evaporated in vacuo and the product purified on silica gelcolumn and eluted with (DCM:MeOH) to yield the compound 6. Purity>93%,ESI m/z 268.40 (MH⁺).

N-(cyclopropylmethyl)-6-phenyl-5-(pyridin-4-yl)pyridazin-3-amine:Compound 6 is aminated using 9 equiv of cyclopropylmethanamine in1-butanol at 130° C. in a pressure vessel for 21 hrs, then the reactionmixture concentrated under reduced pressure, ethylacetate added, and themixture dried over anhydrous sodium sulphate. The crude product ispurified by flash column chromatography (dichloromethane:MeOH 70:30)gives the product. Purity>98%, ESI m/z 302.37 (MH⁺), M.P. 198.7-199.5°C.

1-(2-(6-phenyl-5-(pyridin-4-yl)pyridazin-3-ylamino)ethyl)imidazolidin-2-one:Compound 6 is aminated using 6 equiv of1-(2-aminoethyl)imidazolidin-2-one in 1-butanol at 130° C. in a pressurevessel for 24 hrs, the mixture concentrated under reduced pressure,ethylacetate added and washed with water, then dried over anhydroussodium sulphate. The crude product is purified by recrystallization inmethanol/hexane. Purity>98%, ESI m/z 361.63, (MH⁺), M.P. 205.5-206.7° C.(uncorrected).

Various modification and variation of the described methods andcompositions of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the relevant fieldsare intended to be within the scope of the following claims.

1. A composition comprising a compound described by the followingformula:

including salts, and esters thereof; including both R and S enantiomericforms and racemic mixtures thereof; wherein R1 and R2 are independentlyselected from i) R1 is H and R2 is CH3, or ii) R1 is CH3 and R2 is H;wherein R3 is selected from H, CH3,

wherein R5 is H, CH3, a linear or branched, saturated or unsaturated, asubstituted or unsubstituted, aliphatic chain having less than 5carbons; and wherein R7 is selected from the group consisting of H, CH3,


2. The composition of claim 1, wherein said compound is:


3. The composition of claim 1, wherein said compound is: